This proposal describes the genetic analysis of the Gag and Pol proteins of the Moloney murine leukemia virus and of the cellular host proteins with which they interact. We have used the yeast two-hybrid system to identify novel mammalian proteins that bind to various specific domains of the Gag and Pol gene products. We will now focus our ongoing efforts on several of these new targets: the IQGAP proteins, which interact with the MA portion of Gag; two components of the SUMO transferase system, which interact with the CA portion of Gag; and the ribosomal protein S3a, which binds the RNA pseudoknot at the Gag-Pol border. Biochemical methods will be used to confirm and characterize the interactions between the viral and cellular proteins both in vitro and in vivo. To determine the role of these proteins in virus replication, mutations in the viral genes will be identified that specifically abrogate the interaction with the host protein. These mutations will be introduced into the viral genome and the effects on virus replication in cell culture will be determined. Reversion analysis will be used to further define the requirements for the interaction, and to confirm the importance of the interaction for virus replication. Dominant interfering alleles and RNAi methods will be used to document the requirement for the interaction, and to define the stage of the life cycle at which the proteins act. The generality of the results will be tested by examining the effects of the genes on the replication of a panel of retroviruses of other families. These experiments will significantly extend our understanding of retrovirus replication, and may provide important new targets for antiviral intervention.